CRISPR Cell Line Gene Modification & Point Mutations
CRISPR / Cas9 Service
The CRISPR/Cas9 system uses the Cas9 nuclease to facilitate RNA-guided site-specific DNA cleavage. Our system consists of two components:
(1) A mammalian codon-optimized version of the Cas9 protein carrying a nuclear localization signal to ensure nuclear compartmentalization in mammalian cells
(2) Guide RNAs (gRNAs) to direct the Cas9 protein to sequence-specific cleavage the targeted DNA
The advantage of CRISPR/Cas9 over ZFNs or TALENs are scalability and multiplexibility. With CRISPR/Cas9 multiple sites within a mammalian genome can be simultaneously modified, providing a robust, high-throughput approach for gene editing and CRISPR point mutations in a variety of mammalian cells.
CRISPR (Point mutations, Deletions and Small DNA insertions)
We are experts in CRISPRCas9 Service! Applied StemCell is noted in Nature Biotechnology as one of the select companies for CRISPR/Cas9 tools! Monya Baker, "Gene Editing at CRISPR Speed," Nature Biotechnology, 32: 309-312, April 2014.
Applied Stem Cell uses licensed CRISPRCas9 technology licensed from the Broad Institute. Our proprietary algorithm for designing gRNA has been successful in a number of cancer cell lines, primary cells, induced pluripotent stem cells, as well as in animal models. We use a modified Cas9 nickase to minimize off-target effects and all our gRNAs are functionally validated in vitro before being used to create your cell line. Since Applied Stem Cell started out as a company focused on induced pluripotent stem cells (iPSCs), we have extensive experience amd capabilities for correcting mutations in disease-model iPSCs using CRISPR/ Cas9.
|CRISPR/Cas9 Cell Line Service Timeline|
|1. Targeting DNA Vector Creation||6-14 weeks||Biweekly updates throughout service|
|gRNA Design and Construction (2-4 gRNAs)||2 weeks|
|gRNA in vitro Functional Validation (2-4 gRNAs)||2-4 weeks|
|Donor DNA Construction (knock-in or point mutations)||2-4 weeks|
|2. Cell Culture, Transfection, Optimization||2-4 weeks|
|3. Cell Culture, Transfection/ Electroporation, Selection, Screening, and Clone Confirmation by PCR or Sequencing||7-10 weeks|
|4. Cell Expansion and Cryopreservation||1-2 weeks||Genetically Engineered Cell-Line, 2 vials (2x105 cells/vial)|
We can use either transfected plasmids or oligos to create your cell line. For difficult-to-transfect cell lines, we use electroporation to introduce the gene modification. For Knock-in cell lines, the donor DNA contains either a fluorescent marker (such as GFP) or an antibiotic resistance gene (such as puromycin) for selection.
FAQ for CRISPR Genome Editing Services
1.What genome editing method do you use in your Cell Line service?
For knockout, point mutation, and DNA insertion (small and large DNA insertion): CRISPR/Cas9. For knock-in of DNA fragments in a genomic safe harbor locus, e.g. Rosa26, AAVS1, H11: TARGATT™.
2. Is there a size limit on DNA to be inserted into the genome (CRISPR Knockin)?
For CRISPR/Cas9, we can insert fragements up to 9kb in cell lines with drug selection. For TARGATT™, we can insert fragments up to 20kb.
3. Have you encountered difficulties in genome editing in cell lines?
Typical challenges are:
- The promoter driving Cas9 expression needs to be tested and validated in different cell lines.
- DNA repair system is not active in the cell line.
- Cells are not clonable.
- Low transfection efficiency.
- Abnormal karyotype (cancer cell lines).
- Target region contains repeats and no good gRNAs can be found.
It is important to pre-evaluate all new cell lines for copy number of the target gene, clonability and transfection efficiency of the cell line, and CRISPR vector promoters in order to ensure the success of the project.
4. How many guide RNAs do you typically design for a CRISPR Genome Editing Service?
We start with 2 gRNAs and validate them. If no active gRNA is identified, we do another 2 gRNAs. In most projects, two rounds of testing are sufficient.
5. Can you provide off-target analysis report?
Yes, upon customer’s request.
6. Are you currently providing gene editing service to biotech/pharmatheutical companies as well?
7.Can we have a confidentiality disclosure agreement (CDA) before disclosing my project details?
8.What is the final deliverable product?
We ship at least 2 vials, each at 0.5x10^6 cells/vial, cryopreserved cells per clone with a report of the project. Additional clone(s) and vial(s) are available upon request. For fee-for service projects, you can also have the CRISPR vectors upon request.
Knock-out Case Study #1. Multi-gene knock-outs in human cancer cells.
Surveyor assays and sequencing results showed that a stable homozygous mutant cell line with a two base pair deletion in clone X was successfully produced, causing a frame shift in gene A.
Sequence Data: Final clone X (homozygous) and WT, both forward and reverse sequence of Gene A.
Raw sequence data: Data shows reverse sequence results. Black box highlights the two base pairs present in the WT which are absent in clone X.
Knock-out Case Study #2: Precise deletion of a specific fragment from the target gene in a cancer cell line.
Two gRNA were designed to delete a specific fragment. Both homozygous and heterozygous clones were identified after screening and sequence verification. This example shows that CRISPR/Cas9 can be used for precise deletion.
Knock-in Case Study #3: Point mutation knock-in in mouse fibroblasts.
A Cas9/gRNA-expressing vector was used to introduce a cut at the target site. A single-stranded oligonucleotide (ssODN) was used as a donor for homology-directed repair (HDR) at the cut site introduced by Cas9. Sequence data indicate a two nucleotide change of “CC” to “AG”.
Site-Specific Knock-In of reporter with mCherry
|Delivered Cell Line Engineering Projects (selected)|
|DLD-1 (Colon Cancer)||Human|
|HCT116 (Colon Cancer)||Human|
|HT29 (Colon Cancer)||Human|
|RKO (Colon cancer)||Human|
|HeLa (Cervical cancer)||Human|
|Breast Cancer Cells||Mouse|
|iPSC (various from disease/healthy)||Human|
|HaCaT (Immortal Keratinocyte)||Human|
|Immortalized Bronchial Epithelial Cell Line||Human|
|Immortalized Fibroblast Puromycin Resistant Line||Human|
|Mammary Gland Epithelial||Human|
|Follicular thyroid cell line||Rat|
|CHO (Hamster Ovary)||Chinese Hamster|
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Let our Experts Create a Cell Line for You, or Do It Yourself!
In addition to our full service plan, you can purchase the kits to make a cell line model yourself.
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